Solvent-Free Biodiesel Production Catalyzed by Crude Lipase Powder

Article pubs.acs.org/JAFC

Solvent-Free Biodiesel Production Catalyzed by Crude Lipase Powder from Seeds: Effects of Alcohol Polarity, Glycerol, and Thermodynamic Water Activity Paul Alain Nanssou Kouteu,†,‡ Joel̈ Blin,†,§ Bruno Baréa,§ Nathalie Barouh,§ and Pierre Villeneuve*,§ Institut International d’Ingénierie de l’Eau et de l’Environnement (2iE), Laboratoire Biomasse Énergie et Biocarburants (LBEB), Rue de la Science, 01 BP 594, Ouagadougou 01, Burkina Faso ‡ Montpellier SupAgro, UMR 1208 Ingénierie des Agro-polymères et Technologies Émergentes, 2 Place Viala, F-34060 Montpellier, France § Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), 73 rue Jean-François Breton, 34393 Cedex 5 Montpellier, France †

ABSTRACT: The aim of this work was to evaluate the potential of crude lipase powders made from Adansonia grandidieri and Jatropha mahafalensis seeds for the synthesis of fatty acid alkyl esters in a solvent-free system. The influence of the nature of the alcohol, the amount of glycerol, and hydration of the powder was investigated. Results showed that the activity of these crude lipase powders was inversely proportional to the alcohol polarity and the amount of the glycerol in the reaction medium. To ensure optimum activity, A. grandidieri and J. mahafalensis powders must be conditioned to a water activity of 0.33 and 0.66. To obtain a fatty acid ethyl ester yield greater than 95% with A. grandidieri, ethanol should be introduced at an amount corresponding to a triacylglycerol to ethanol molar ratio of 2:1 every 15 h for 96 h and use 25% of preconditioned crude lipase powders (2 additions of 12.5%). KEYWORDS: fatty acid ethyl ester, transesterification, thermodynamic water activity, A. grandidieri, J. mahafalensis, crude lipase powder

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INTRODUCTION Declining petroleum reserves and environmental problems associated with their use has led to the search for alternative fuels for internal combustion engines.1 In this context, biodiesel has received considerable attention due to its biodegradability, nontoxicity, and physical−chemical characteristics, which resemble those of diesel.2−4 Biodiesel can be used as a partial substitute in a mixture or completely replace diesel through a minor modification of the engine.3,5 Biodiesel, a mixture of fatty acid alkyl ester (FAAE), is generally obtained by transesterification of triacylglycerol with an alcohol in the presence of a chemical or enzymatic catalyst. Lipases, triacylglycerol acylhydrolase (E.C.3.1.1.3), are among the enzymes that are mostly used to catalyze transesterification.3,5 They are widely distributed in nature (plants, animals, microorganisms), where they play an important physiological role in lipid metabolism. There are several advantages using enzyme catalysis rather than chemical catalysis for the production of biodiesel: (a) the ability of the enzyme to work in different medium including biphasic or monophasic systems (in the presence of hydrophobic or hydrophilic solvent), (b) the use of a variety of alcohols (methanol, ethanol, propanol, butanol, isopropanol, etc.) and lipid substrates (refined or nonrefined oil, waste cooking oils, free fatty acid, etc.), (c) the absence of side reactions and the ability to work under milder conditions thereby minimizing energy consumption, and (d) easy recovery of glycerol and the high purity of the final product.5−10 However, the use of lipases for the production of biodiesel is limited by their cost and © 2017 American Chemical Society

potential inhibition by alcohols (glycerol, methanol, ethanol, etc.).4,6,11 On the other hand, these limits can be overcome by using a solvent or by stepwise addition of the alcohol to the reaction medium.5,6,8,9,11 To date, the great majority of lipases used in biodiesel processes are of microbial origin5,6 and produce satisfactory yields (90−95%). Less attention has been paid to the use of plant lipases in these processes.6 Their advantages, which include low cost, and that they can be used as biocatalyst with only partial purification, make lipases of plant origin a promising alternative to the use of microbial enzymes and conventional chemical catalysts.2,12−14 In a recent study, it was possible to use crude lipase powder (CLP) made from germinated seeds of Adansonia grandidieri and Jatropha mahafalensis, delipidated or not, to catalyze the synthesis of fatty acid alkyl ester (FAAE) from sunflower oil with ethanol as the acyl acceptor.15 Compared with methanol mostly used for biodiesel production, ethanol is less toxic and easy to obtain by fermenting sugar rich biomasses, thereby giving a completely agricultural fuel.2,16 The activity of these powders was influenced by the nature of solvent used when delipidation was required, the TAG to ethanol molar ratio, and the reaction temperature. The optimal activity of the powder was obtained when hexane was used as solvent for the Received: Revised: Accepted: Published: 8683

July 6, 2017 September 5, 2017 September 7, 2017 September 7, 2017 DOI: 10.1021/acs.jafc.7b03094 J. Agric. Food Chem. 2017, 65, 8683−8690

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Journal of Agricultural and Food Chemistry

determined by comparing the mass obtained at the different stages with the mass of the dry sample. The isotherms were obtained by plotting the water content of the CLP versus water activity (aw = HR/ 100). Effect of Glycerol on the Transesterification Activity of Crude Lipase Powders. The reactions were carried out for 72 h in an incubator (IKA KS 4000i control, Legallais, France) at 30 °C with a triacylglycerol to ethanol molar ratio of 2:1 with CLP made from J. mahafalensis seeds. With CLP made from A. grandidieri seeds, the reactions were performed at 40 °C with a triacylglycerol to ethanol molar ratio of 1:1. Different amounts of glycerol (1.7%, 3.4%, 5.1%, 6.8%, and 10%, w/w of oil) were introduced into the medium. After orbital stirring at 250 rpm for 1 h, 12.5% (w/w of oil) CLP was added to the medium to initiate the reaction. Equilibration of Crude Lipase Powders at the Desired Water Activity. The CLPs were incubated for at least 2 weeks at 25 °C in vacuum desiccators containing different saturated salt solutions in order to fix their water activity with that of the reference salts. The different salts used were P2O5 (aw < 0.042), LiCl (aw = 0.11), CH3COOK (aw = 0.22), MgCl2 (aw = 0.33), K2CO3 (aw = 0.44), Mg(NO3)2 (aw = 0.55), NaCl (aw = 0.75), and KNO3 (aw = 0.95). The TWA of the incubated CLPs was then checked using a water activity meter (Tripette and Renaud, Aqualab Type 3TE, France) before the CLPs were added to the reaction medium. Optimization of Transesterification Reaction by Stepwise Addition of Ethanol. All the reactions were carried out in an incubator (IKA KS 4000i control, Legallais, France) at 40 °C under orbital stirring at 250 rpm with 4 g of sunflower oil and 12.5% (w/w of oil) CLP. Four different strategies of stepwise addition of ethanol in the reaction medium were tested.

delipidation of both powders, and when the TAG to ethanol molar ratio was 2:1 and temperature 30 °C for CLP made from J. mahafalensis seeds and 1:1 and 40 °C for CLP made from A. grandidieri seeds. Several other factors not investigated in previous works including alcohol polarity, glycerol, and thermodynamic water activity (TWA) are crucial to control lipase activity,4,5,17,18 and analysis of their influence is needed to improve the synthesis of fatty acid ethyl ester (FAEE) yield. The aim of the present study was thus to analyze the influence of alcohol polarity, glycerol, and thermodynamic water activity. The CLP with the best characteristics was then chosen to explore different stepwise additions of ethanol in the medium to obtain the complete conversion of TAG into FAEE.

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MATERIALS AND METHODS

Materials. Sunflower oil was purchased in a local supermarket (Montpellier, France). J. mahafalensis and A. grandidieri seeds, harvested in May 2014, were obtained from PhileoL in the region of Androy (Madagascar). High-performance thin-layer chromatography silica plates (HPTLC, 20 × 10 cm silica gel Plate 60) were purchased from Merck (Darmstadt, Germany). All products and chemical solvents used were of analytical grade or higher and were purchased from Sigma-Aldrich (Saint Quentin, France). Preparation of Crude Lipase Powders. The seeds were first washed with water in a beaker. J. mahafalensis seeds were then soaked in water at room temperature for 6 h, whereas A. grandidieri seeds were soaked for 2 days because of the hardness of their coats. One kilogram of soaked seeds was spread out in one layer between two moistened sheets of paper in a thermostatic oven (25 °C) (Fischer Scientific, Memmert, HPP108L, Germany) with controlled humidity (90%). After 4 days of germination, the seeds were husked by hand, crushed (Waring, France), and then dried at 35 °C in a vacuum oven (Bioblock Scientific, 45001, France) for 24 h. The resulting powders were delipidated with hexane for 8 h using a Soxhlet extractor equipped with a cooling system according to the French standard NF V 03-924. To remove any remaining traces of hexane, the powders were dried at room temperature overnight. Finally, the powders were ground in a blender (Waring, France), sieved to obtain a particle size less than 635 μm, and stored at 4 °C until use. Transesterification Reactions with Various Alcohols. The experiments were performed with a triacylglycerol to alcohol (ethanol, propanol, butanol, pentanol, and hexanol) molar ratio of 1:3 in a solvent-free medium for 72 h in an incubator (IKA KS 4000i control, Legallais, France) with orbital stirring (250 rpm) at 30 °C for CLP made from J. mahafalensis and at 40 °C for A. grandidieri seeds. The reactions were initiated by adding 12.5% (w/w of the oil) CLP. At the end of the reaction, the resulting FAAE were quantified by highperformance thin-layer chromatography (HPTLC) combined with densitometric measurements. Measurement of CLP Water Sorption and Desorption Isotherms. The sorption and desorption isotherms of the CLP from J. mahafalensis and A. grandidieri seeds were determined by using dynamic vapor sorption (DVS, Surface Measurement Systems Ltd., London, U.K.). The system consists of an ultrasensitive Khan microbalance (sensitivity 0.1 μg) placed inside a temperature controlled hermetically sealed chamber at a constant temperature of 20 °C. The measurements were made on 15 mg of CLP previously dehydrated under P2O5 at room temperature in a desiccator for 1 week. The relative humidity of the DVS chamber was regulated by an air flow comprising a mixture of a dry and saturated vapor gas in the selected proportions. The equipment was set up to generate 11 relative humidity levels ranging from 5% to 95% for sorption and from 95% to 5% for desorption. An equilibrium criterion was defined corresponding to a variation in mass over time (dm/dt) of 0.002% min−1. The shift from one relative humidity to the other occurred when the variation in mass of the sample was less than 0.002% min−1. The change in mass caused by the sorption or desorption of the water molecule was recorded by the microbalance. The water content of the sample was

• Three additions of 0.21 g (corresponding to an oil to ethanol molar ratio of 1:1) of ethanol at 0, 30, and 60 h. • Three additions of 0.21 g (corresponding to an oil to ethanol molar ratio of 1:1) of ethanol at 0, 30, and 60 h and the addition of a new CLP (12.5%, w/w of oil) at 30 h. • Four additions of ethanol: two additions of 0.21 g at 0 and 30 h and two additions of 0.105 g (corresponding to an oil to ethanol molar ratio of 2:1) at 60 and 75 h. A new CLP (12.5%, w/w of oil) was added at 30 h. • Six additions of 0.105 g of ethanol at 0, 15, 30, 45, 60, 75 h and the addition of a new CLP (12.5%, w/w of oil) at 30 h. Samples of the different reaction media were taken periodically and analyzed by HPLTC coupled with densitometric measurements. High-Performance Thin-Layer Chromatography Coupled with Densitometric Analysis. The FAEE and free fatty acids (FFA) in the reaction medium were quantified by HPTLC coupled with densitometric measurements according to the protocol established by Moussavou et al.2 with some modifications. Different amounts of standards (ethyl esters or fatty acids from sunflower oil) and the samples to be analyzed were deposited on a silica plate (HPTLC, 20 × 10 cm, Merck) using an automatic ATS4 (Camag, Muttenz, Switzerland) under nitrogen atmosphere. Elution was carried out with a hexane/diethyl ether/acetic acid mixture (80:20:2, v/v/v). The different compounds were revealed by immersion in a copper sulfate:phosphoric acid:methanol:water solution (10:8:5:78, v/v/v/v) followed by carbonization of the plates at 180 °C for 10 min. The amounts of different lipid species were determined by scanning the plates at 550 with a TLC3 scanner (CAMAG, Muttenz, Switzerland). The amount (μg) of FAEE or FFA in the different reaction media was derived from the calibration curve. All the analyses were performed in triplicate, and results are given as mean ± standard deviation. Yields were calculated based on the limiting substrate: FAEE yield (%) =

nFAEE × 100 nlimiting substrate

where nFAEE and nlimiting subtrate are the number of moles of FAEE and limiting substrate, respectively. 8684

DOI: 10.1021/acs.jafc.7b03094 J. Agric. Food Chem. 2017, 65, 8683−8690

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Journal of Agricultural and Food Chemistry Statistical Analyses. The results were expressed as the mean ± standard deviations of at least three repetitions. Using Statgraphics Centurion XVI (Statpoint Technologies, USA), Student’s t test was performed for the comparison of the means. The significance level α was set to 0.05.

were carried out by adding increasing amounts of glycerol to the reaction medium (1.7%, 3.4%, 5.1%, 6%, 8%, and 10% compared to TAG). The relative activities were then calculated using the ratio between the activities of the lipase in the presence or absence of the glycerol in the reaction medium, as shown in Figure 2.

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RESULTS AND DISCUSSION Influence of Alcohol Polarity on the Transesterification Activity of Crude Lipase Powders Made from J. mahafalensis and A. grandidieri Seeds. Different alcohols are generally used for the production of FAAE. Five primary linear alcohols (ethanol, propanol, butanol, pentanol, and hexanol) were selected to study the influence of the alcohol polarity on the capacity of the CLP made from A. grandidieri and J. mahafalensis seeds to synthesize the corresponding esters. A triacylglycerol to alcohol molar ratio of 1:3 was used to compare possible inhibition of these lipases at this ratio since previous work15 showed that the activities obtained with ethanol were almost zero at this ratio. Whatever the alcohol used, the highest yields were obtained with the CLP made from A. grandidieri seeds (Figure 1). With

Figure 2. Effect of added glycerol on transesterification activity of CLP made from A. grandidieri and J. mahafalensis seeds. Reaction conditions: reaction time 72 h, 12.5% (w/w of oil) of CLP, 30 °C (triacylglycerol to ethanol molar ratio 2:1), and 40 °C (triacylglycerol to ethanol molar ratio 1:1) for the CLP made from J. mahafalensis and A. grandidieri seeds, respectively.

Whatever the CLP, the relative activity decreased with the larger amounts of glycerol added to the medium (Figure 2). At 10% glycerol, theoretical amount formed after the complete conversion of triacylglycerol, the relative activities of the two CLPs were less than 20%, indicating their inhibition by the glycerol. This phenomenon has already been described by several teams,4,27 including that of Kawakami et al.,25 who reported a 30% decrease in Burkholderia cepacia lipase activity following the addition of 10% glycerol to the medium during methanolysis of J. curcas oil. According to Pedersen et al.28 and Xu et al.,4 glycerol may interact specifically with the enzyme, as a substrate or an inhibitor, or its physical−chemical properties (viscosity, density, etc.) may influence the mixing of the reaction medium. Of the two CLPs tested under these conditions, the CLP made from A. grandidieri seeds was the most sensitive to glycerol. With 1.6% glycerol in the medium (the smallest amount tested), it displayed only 27% of its activity while that of J. mahafalensis was still 50%. This difference in behavior could be explained by the properties of the CLP powders. Xu et al.4 tested four supports and showed that the most hydrophilic supports are those that adsorb a greater amount of glycerol. The CLP made from A. grandidieri seeds may therefore be more hydrophilic than that from J. mahafalensis. Effect of Initial Thermodynamic Water Activity (TWA) on FAEE Synthesis Catalyzed by Crude Lipase Powders from J. mahafalensis and A. grandidieri Seeds. The catalytic activity of the lipases in transesterification in a nonaqueous medium with anhydrous substrates is highly influenced by the TWA of the lipase.29−32 To assess the effect of the initial TWA of the CLP on their transesterification activity, the reactions were carried out under the optimum synthesis conditions that had previously been determined for each of the powders15 (30 °C, triacylglycerol to ethanol molar

Figure 1. Fatty acid ester yields obtained during transesterification of sunflower oil with various alcohols catalyzed by CLP made from A. grandidieri and J. mahafalensis seeds. Reaction conditions: TAG to alcohol molar ratio 3:1, reaction time 72 h, 12.5% (w/w of oil) of CLP, 30 and 40 °C for the CLP made from A. grandidieri and J. mahafalensis seeds, respectively.

this powder, FAAE yields were 2%, 26%, 29%, 36%, and 53% with ethanol, propanol, butanol, pentanol, and hexanol, respectively. Thus, the lower the alcohol polarity, the higher the yield of FAAE. The same trend was observed with CLP made from J. mahafalensis seeds (Figure 1). This behavior is similar to that reported for other plant lipases.6,17,19 There are two possible reasons for the low yield observed with ethanol. One is that lipase is inactivated by contact with ethanol, which is insoluble at these stoichiometric proportions (ratio 1:3), unlike the other alcohols.20,21 The other is that the energy released by ethanol when bound to the active site of the enzyme is not sufficient to allow a change in the conformation of the lipase.22,23 Effect of Glycerol on FAEE Synthesis Catalyzed by Crude Lipase Powders Made from J. mahafalensis and A. grandidieri Seeds. The presence of glycerol in the medium, coproduct from the transesterification reaction of triacylglycerol with alcohols, is known to alter lipase activity during the reaction.24−26 In order to assess its effect on the activity of CLP made from J. mahafalensis and A. grandidieri seeds, experiments 8685


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Journal of Agricultural and Food Chemistry ratio 2:1 for J. mahafalensis, and 40 °C, triacylglycerol to ethanol molar ratio 1:1 for A. grandidieri) and by conditioning them to a TWA varying from 0.04 to 0.8. Figure 3 shows the yields of FAEE over time with the different powders incubated at a fixed water activity.

The activity (initial velocity and yield) of the two CLPs in ethanolic transesterification is therefore correlated with their initial TWA. According to this dependence, Xia et al.33 divided the lipases into three groups. The first group included lipases whose activity was optimal at low TWA, while the second and third groups contained lipases whose activity was optimal at intermediate and high TWA, respectively. CLPs made from A. grandidieri and J. mahafalensis seeds are therefore close to the first and second groups, respectively. Water Sorption and Desorption Isotherms of Crude Lipase Powders Made from J. mahafalensis and A. grandidieri Seeds. The sorption and desorption isotherms reveal changes in water content as a function of the TWA of the CLP. On the one hand, they were identified to estimate the amount of water contained in the powder as a result of its incubation at a precise level of water activity, and on the other hand to correlate the effect of the activity of water on their transesterification activity according to different states of hydration of the CLP. The sorption/desorption isotherms of the CLP made from A. grandidieri and J. mahafalensis seeds are sigmoidal in shape and are perfectly superposable and symmetrical (Figure 4). The hysteresis phenomenon is practically nonexistent, meaning that, at a given water activity, the quantity of water absorbed by the

Figure 3. Influence of initial thermodynamic water activity on the FAEE yield. Reaction conditions: reaction time 72 h, 12.5% (w/w of oil) CLP, (A) J. mahafalensis (30 °C, triacylglycerol to ethanol molar ratio 2:1) and (B) A. grandidieri (40 °C, triacylglycerol to ethanol molar ratio 1:1).

With the CLP made from A. grandidieri seeds, the highest initial velocity (19.4 nmol·min−1·mg−1 protein) was obtained with a water activity value of 0.33, which was not significantly different (p ≤ 0.05) from that obtained with 0.04, 0.15, and 0.24. The highest yield (99%) was also obtained at this water activity. There was not a significant (p ≤ 0.05) increase of FAEE in the medium at 30 h with CLP which had a water activity less than or equal to 0.42, whereas with the other incubated CLP it was reached in 48 h. The CLP made from J. mahafalensis needed to be incubated at a high TWA (0.66) to have an initial rate (6.3 nmol·min−1· mg−1 protein) and a yield (80%) at their maximum. The equilibrium with this CLP was reached in 48 h, meaning that there was not a significant (p ≤ 0.05) increase of ethyl ester after this time, unlike the other CLP, which reached equilibrium in 30 h, but with FAEE yields of less than 35%.

Figure 4. Water sorption and desorption isotherm of CLP made from J. mahafalensis (A) and A. grandidieri seeds (B). 8686


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Journal of Agricultural and Food Chemistry different powders does not significantly differ from the quantity desorbed. Similar behavior has been reported for Babaco latex lipase powders.29 The sorption isotherms of the CLPs from A. grandidieri and J. mahafalensis seeds show a linear uptake of water molecules up to a TWA of 0.5 (Figure 4.). Beyond this value, adsorption becomes exponential. The linear part was divided into two zones. The first zone, between a TWA of 0 and 0.2, is hypothesized to represent the formation of a molecular monolayer of water bound to the surface of the product through the establishment of the van der Waals forces between the hydrophilic groups of CLP and the water molecule. The second linear zone, between a TWA of 0.2 and 0.5, is hypothesized to correspond to the formation of other layers of water on the preformed monolayer. The exponential zone is hypothesized to correspond to very weakly bound water present in the liquid state in the pores of the powder. The water contents of the different powders at the last point of this exponential phase (aw = 0.95) were 40.1% and 51.2% for the CLP made from J. mahafalensis and A. grandidieri seeds, respectively (Figure 4), showing that the latter absorbs more water. CLP made from A. grandidieri seeds is therefore the most hydrophilic of the two CLP. These observations confirm those reported during the study of the influence of glycerol, in which it was concluded that the greatest sensitivity of the CLP made from A. grandidieri seeds to glycerol is probably linked to the fact that this powder is the most hydrophilic. Relationship between Ethanolic Transesterification Activity and the State of Hydration of the Crude Lipase Powders Made from J. mahafalensis and A. grandidieri Seeds. The sorption isotherm of the two CLPs showed that they adsorb large amounts of water, which can lead to a competitive hydrolysis reaction during the transesterification reaction. In order to evaluate the effect of this phenomenon on the transesterification yields, the FFA produced during this reaction (30 °C, triacylglycerol to ethanol molar ratio 2:1 for J. mahafalensis, and 40 °C, triacylglycerol to ethanol molar ratio 1:1 for A. grandidieri) were quantified. Figure 5 shows the water content of the CLP and the FAEE and FFA yields as functions of the initial water activity of the powders. It is known that a minimum level of hydration is required for a lipase to have an optimum active spatial configuration.30 The optimal catalytic activity of CLP made from A. grandidieri seeds was obtained when the lipase was previously incubated at a TWA of 0.33, i.e., a water content of 3.5% based on the sorption isotherm (Figure 5B). This optimum level of hydration appears in the linear part of the curve of the sorption isotherm, that is to say, when the water molecules form one or more monolayers with the protein network. Other plant lipases, such as that obtained from Babaco latex,29 also require a monolayer of water for optimal activity. The hydration of the CLP made from A. grandidieri seeds between 3.5% and 6.1% (corresponding to a TWA of 0.52) led to competition between the synthesis reaction and that of hydrolysis (Figure 5B). The decrease in the transesterification activity of the CLP made from A. grandidieri seeds in this interval (0.33 < aw < 0.52) is hypothesized to be due to this competition. It is known that the ethanolic transesterification mechanism occurs through the formation of an acyl−enzyme transient complex that then reacts with the alcohol. In this part of the sorption isotherm (0.33 to 0.52), the water molecules are linked to the protein network or to the vegetable matrix with low energy bonds and can react with this complex to the detriment of the alcohol,

Figure 5. Influence of the aqueous state of CLP made from J. mahafalensis (A) and A. grandidieri (B) seeds on their transesterification activity.

hence promoting the hydrolysis reaction. With the CLP incubated at 0.66 and 0.8 (corresponding to a water content of 10.8% and 18%, respectively), loss of activity (hydrolysis and transesterification) was observed (Figure 5B), which may be due to modification of the native configuration of the protein. Indeed, the CLPs incubated at these TWA, particularly when incubated at 0.8, lost their powdered state and formed a gel. This observation is in agreement with that made in other studies which have demonstrated agglomeration of the catalyst at high levels of hydration resulting in a reduction in catalyst activity.31,32,34 The optimum state of hydration of CLP made from J. mahafalensis seeds (10.7%) corresponds to the beginning of the exponential phase of the sorption isotherm when the water is present in the unbound form and plays the role of solvent (Figure 5A). The presence of free water around the protein is hypothesized to reduce the denaturing effect of ethanol on the lipase, thus allowing better expression of its activity. Like A. grandidieri, the loss of activity (in hydrolysis and transesterification) of J. mahafalensis for TWA greater than 0.66 (Figure 5A) may be explained by the formation of a gel, which means that they cannot be used as catalysts. Whatever the CLP used, loss of activity was observed when the powders were incubated at a TWA of 0.8. To check whether a reduction in the water content of these powders enabled resumption of activity, the different powders incubated at a TWA of 0.8 were dehydrated with P2O5 (aw < 0.04) and their activity was reassessed. The activities obtained in this way 8687


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Figure 6. Time course profiles of different ethanol feeding strategies during transesterification of sunflower oil catalyzed by CLP made from A. grandidieri seeds. Reaction conditions: 40 °C, 12.5% (w/w of oil), 4.56 mmol of sunflower oil. (A) 1 equiv of ethanol (corresponding to a triacylglycerol to ethanol molar ratio of 1) was added at 0, 30, and 60 h. (B) 1 equiv of ethanol was added at 0, 30, and 60 h; and 12.5% (w/w of oil) of a fresh CLP was added at 30 h. (C) 1 equiv of ethanol was added at 0 and 30 h; 0.5 equiv of ethanol was added at 0, 30, and 60 h; and 12.5% (w/ w of oil) of a fresh CLP was added at 30 h. (D) 0.5 equiv of ethanol was added at 0, 15, 30, 45, 60, and 75 h; and 12.5% (w/w of oil) of a fresh CLP was added at 30 h. (↓) Addition of ethanol in the medium. (↑) Addition of a fresh CLP in the medium.

when more than one molar equivalent of ethanol is present in the medium. One of the methods proposed by many authors11,21 to avoid this phenomenon and to allow complete conversion of TAG is stepwise addition of alcohol. Equilibrium was reached in 30 h of reaction during transesterification with the CLP made from A. grandidieri seeds conditioned to its optimal water activity (0.33) (Figure 3). Based on these results, four strategies for stepwise addition of ethanol were tested (Figure 6). In the first step, one molar equivalent of ethanol (corresponding to a TAG to ethanol molar ratio of 1:1) was added at 0, 30, and 60 h. As shown in Figure 6A, a FAEE yield of 40% was obtained after 96 h of reaction. The last two additions (30 and 60 h) only led to an increase of 10% compared to the first phase (30%), indicating a decrease in lipase activity after the first 30 h of reaction. This result implies that, after 30 h of reaction, the lipase needs more than 30 h for the conversion of a corresponding quantity of triacylglycerol to FAEE at a triacylglycerol to ethanol molar ratio of 1:1. Since

were lower than those of CLPs initially conditioned to this TWA (aw < 0.04). Thus, the loss of activity following incubation of the CLP at 0.8 appears to be irreversible. These results underline the importance of the conditions in which the powders are stored, particularly the importance of controlling their capacity to absorb water in their matrix during storage, as this can alter the synthesis activity of the CLP during transesterification. The study of all these parameters showed that the CLP from A. grandidieri seeds had the best characteristics for nonaqueous synthesis of FAEE. The rest of this study was therefore conducted using this CLP to develop a strategy that would allow complete conversion of triacylglycerol into FAEE. Effect of the Stepwise Addition of Ethanol on Fatty Acid Ethyl Ester Synthesis Catalyzed by Crude Lipase Powder Made from A. grandidieri Seeds. For complete conversion of TAG to FAEE, stoichiometrically, at least 3 mol of ethanol per mole of TAG would be required. However, the activity of the CLP made from A. grandidieri seeds is inhibited 8688


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ethanol is not completely consumed after 60 h of reaction, the addition of a new ethanol equivalent at this reaction time is therefore hypothesized to result in inhibition of the lipase. Given the sensitivity of the CLP made from A. grandidieri seeds to glycerol, the decrease in activity observed after 30 h of reaction could also be due to the presence of the glycerol formed. Considering this decrease in activity, the second strategy consisted of adding a new quantity of CLP after 30 h of reaction in addition to the stepwise addition of ethanol (1 equiv at 0, 30, and 60 h). This technique led to a FAEE yield of 61% (Figure 6B). However, the kinetics of the second strategy revealed behavior similar to that observed in the first strategy after the addition of an ethanol equivalent at 60 h, thus confirming that, after 30 h of reaction, the CLP loses its activity. Taking this effect into account, the last fraction of ethanol was added in two halves, half at 60 h and half at 75 h (3rd strategy). The fourth strategy consisted of reducing all ethanol additions to 1/2 equiv every 15 h. FAEE yields of 91% and 96% were obtained in 96 h of reaction with the third and fourth strategies, respectively (Figure 6C,D). The stability of the powder after 30 h of reaction was thus improved by fractionating the ethanol additions from one equivalent to a half equivalent. The last strategy, corresponding to the addition of 1/2 equiv of ethanol every 15 h, combined with the addition of a new CLP at 30 h, proved to be the most effective. However, it should be kept in mind that this strategy has the disadvantage of consuming about 25% of the total biocatalyst. In view of the improvement of the FAEE yield using stepwise addition of ethanol, the first reason given to explain the low yield observed with ethanol during the study of the alcohol polarity on the activity of crude lipase powder is surely much more probable than the second. In conclusion, this study emphasizes the importance of a certain number of parameters in the activity of the CLPs made from J. mahafalensis and A. grandidieri seeds. The lipase activity of the two powders depends on the polarity of the alcohol and on the amount of glycerol present in the medium. The CLP made from A. grandidieri seeds proved to be the most sensitive to glycerol, and this behavior was attributed to its hydrophilicity. The determination of sorption isotherms appeared to support this hypothesis. This determination also highlighted the importance of controlling TWA to ensure optimal activity of the biocatalyst. Finally, the best FAEE yield (96%) was obtained with the CLP made from A. grandidieri seeds after 96 h of reaction with the strategy of stepwise additions of alcohol. The CLP, particularly that made from A. grandidieri seeds, without purification prior to use, is thus an alternative to the widely used but more expensive enzyme catalysts. For a successful commercial scale implementation of this CLP, a technoeconomic analysis has to be done.

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The authors thank the Agence Universitaire de la Francophonie (AUF) and the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) for grants to P.A.N.K. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was conducted in the framework of the PRONOVABIO project, with the financial assistance of the European Union. The contents of this publication are the sole responsibility of the partners and can under no circumstances be regarded as reflecting the position of the European Union. The authors would also like to acknowledge PhileoL for providing the seeds used in this study.

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ABBREVIATIONS USED CLP, crude lipase powder; FAAE, fatty acid alkyl esters; FAEE, fatty acid ethyl ester; FFA, free fatty acid; TWA, thermodynamic water activity; DVS, dynamic vapor sorption; HPTLC, high performance thin-layer chromatography

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*UMR 1208 IATE Bâtiment 33, 2 Place Viala, 34060 Montpellier, France. Phone: +33 (0) 4 99 61 20 29. E-mail: [email protected]. ORCID

Pierre Villeneuve: 0000-0003-1685-1494 8689


Article

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Solvent-Free Biodiesel Production Catalyzed by Crude Lipase Powder

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